The Ddz group was introduced by Birr in 1972.sup.6 as an N.sup..alpha. -urethane protecting group labile to dilute solutions of TFA in CH.sub.2 Cl.sub.2. The use of this group allowed a milder overall procedure for the synthesis of peptides in the solid phase, compared to Boc-based strategies..sup.1-2 The Ddz group is cleaved within 15 minutes on the solid phase in 1-5% TFA (V/V) in CH.sub.2 Cl.sub.2. With smaller peptides, 1% TFA in CH.sub.2 Cl.sub.2 is adequate for quantitative removal of the Ddz group. With longer peptides, 5% TFA in CH.sub.2 Cl.sub.2 is required, due to the larger number of amide bonds relative to the N.sup..alpha. -Ddz group. Amide bonds are able to form hydrogen bonds with the reagent acid, effectively reducing its concentration to a point where cleavage is incomplete.
Birr and coworkers have demonstrated the applicability of Ddz-amino acids to solution and solid-phase peptide synthesis by synthesizing peptide fragments on the solid phase for subsequent fragment condensation in solution. In this manner, the mast-cell-degranulating peptide was synthesized both as the fully-protected peptide.sup.35 and the free peptide..sup.36 Ddz-amino acids were also used to construct a protected insulin A-chain,.sup.40-41 which upon deprotection was combined with natural B-chain from Bovine insulin to yield fully active semisynthetic insulin. Thymosin .alpha..sub.1,.sup.43-45 a 28-amino acid peptide, was synthesized completely in solution from fragment condensations.
Of particular note is the application of Ddz-amino acids to large scale solution-phase synthesis of five fragments of thymosin .alpha..sub.1 and twin .alpha..sub.1 peptides in amounts of 400 g or more..sup.45 In this synthesis, DCC/HOBt mediated couplings were used for the preparation of small peptide fragments that were then condensed using the azide method.
Zanotti and colleagues.sup.48 applied Ddz-amino acids throughout their synthesis of amaninamides, analogs of the highly toxic mushroom amatoxins. Use of Boc-amino acids was not feasible, due to the presence of the acid-labile cysteine(S-trityl) residue, and use of Fmoc-amino acids was prohibited due to the protection of the C-terminal .gamma.-hydroxyamino acid as the lactone, this functionality being sensitive to ring opening by amines. Hence, Birr has established coupling and deprotection for the employment of N.sup..alpha. -Ddz amino acids in both solution and solid phase peptide synthesis.
In previous work,.sup.6 most of the Ddz-amino acids were prepared as their corresponding dicyclohexylamine salts. These salts must be manually liberated and combined with an appropriate acylating agent prior to use in solid phase peptide synthesis.
Active esters (hydroxysuccinimide, nitrophenyl, 2,3,5-trichlorophenyl, or pentachlorophenyl).sup.15-16, 19-20, 49-50 of the acid-labile Bpoc-group have been synthesized, but were shown to be too inefficient for application to solid-phase synthesis. The Pfp and ODhbt esters of N.sup..alpha. -Fmoc amino acids have also been prepared.
Kovacs noted the favorable properties of Pfp esters in his study of N.sup..alpha. -urethane-protected cysteine derivatives,.sup.50 in which Pfp esters were identified as having the highest k.sub.coup /k.sub.rac ratio out of a wide range of active esters. Fmoc amino acid Pfp esters were first prepared in Kisfaludy.sup.51 and were later applied to solid phase synthesis in an Fmoc/polyamide continuous flow system by Atherton and Sheppard..sup.52 Hudson,.sup.53 in a comparison of couplings of active esters, stated that Pfp esters were the most suitable for routine use in SPPS, as they are usually crystalline, are prepared in high yield, and are stable to long periods of storage.
ODhbt esters have generally higher reactivity compared to OPpf esters, though their stability is said to be marginal. The in situ preparation of 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl (ODhbt) esters of certain urethane protected amino acids was disclosed by Konig and Geiger (43), but did not gain favor due to the formation of an o-azidobenzoic acid ester as a by-product of their preparation. Fmoc amino acid ODhbt esters, however, have been prepared as crystalline solids in acceptable yields by Atherton, et al,.sup.54 and their use was demonstrated through the synthesis of the acyl carrier protein decapeptide sequence 65-74 and a nonadecapeptide sequence.
A recent review of solid-phase peptide synthesis is provided in Barany, G. et al..sup.2 This reference is specifically incorporated by reference in its entirety herein to provide details of solid-phase synthetic methods.
The present work relates to Pfp and ODhbt activated esters of N.sup..alpha. -Ddz amino acids, particularly those that are crystalline, which have apparently not been reported previously.